| PART ONE: EXPRESSION OF ENT1IN EPILEPTICPATIENTS AND KINDLED ANIMAL MODELObjective: To observe the expression of ENT1in epileptic temporallobe and time-space expression relationship in lithium chloride-pilocarpinekindled epileptic rats hippocampus.Methods：1. Random samples of15temporal lobe of epileptic patients and15ageand sex matched temporal lobe of trauma patients were selected from ourbrain bank. The ENT1expression in patients brain tissues were detected bywestern blot.2. Adult male Sprague-Dawley rats (around200g) were randomlydivided into normal control group (n=5) and epilepsy group (for6h,24h,72h, and1w, n=5×4). By intraperitoneal injection of lithiumchloride-pilocarpine, epilepsy experimental animal model was established.The ENT1expression in hippocampus of rats were detected by weaternblot，immunohistochemistry and immunofluorescence. Results:1. ENT1protein from individual temporal neocortex of patients withepilepsy(1.18±0.15) were stronger than those in nonepilepticpatients(0.50±0.10)，and there were significant differences（p＜0.01）.2. Hippocampal ENT1protein in epileptic rats6h,24h,72h and1wafter the onset of seizures were stronger than those in normal control rats，and there were significant differences（p＜0.01）. In addition, ENT1wasfound in both neurons and astrocytes cytoplasm by immunohistochemistryand immunofluorescence double labeling technique.Conclusion：The ENT1expression in the brain tissue patients withepilepsy and experimental rat model were increased, which prompted ENT1might be involved in the occurrence and development of epilepsy. PART TWO: FUNCTION DETECTION OF ENT1ONEPILEPTIC SEIZURE IN VIVOObjective: To examine whether inhibition of ENT1in the hippocampusalters epileptic behaviors.Methods：The the ENT1selective inhibitor NBTI (50μM) and itssolvent（DMSO） were injected into the rat hippocampus CA1region bythree-dimensional positioning and microtubule.1．Adult male SD rats were randomly divided into normal control(Control) group （n=9）, solvent control (vehicle) group（n=9） andnitrobenzylthioinosine (NBTI) group（n=9）. Before intraperitoneal injectionof pilocarpine, rats were microtubule injected with normal saline,dimethylsulfoxide and NBTI respectively.2. After intraperitoneal injection of pilocarpine, the latency to firstseizure and Racine score in different time periods were detected.Results: Whose Racine score reached level4and above was consideredas epilepsy. Rats treated with NTB1exhibited significantly longer onsetlatency and less seizure severity with lower Racine score (P <0.01).Conclusion： The effective dose NBTI intervention significantlyprolonged the latency and reduced the intensity of seizures, in other words,the selective inhibition of ENT1function could suppress seizureseffectively. PART THREE: FUNCTION DETECTION OF ENT1ONEPILEPTIC HIPPOCAMPAL BRAIN SLICES IN VITROObjective: To observe epileptic rats hippocampus brain sliceelectrophysiology indexs change and the effect of ENT1selective inhibitorNBTI through the whole cell patch clamp technique, which coulddemonstrate the function of ENT1in epileptic rats hippocampus brain slicesin vitro.Methods：Preparation of adult rat hippocampal slices were randomlyselected from normal adult rats and successful modeling epileptic rats(Based on the results of first part of the experiment, the ENT1expressionpeak time point of24h was selected). Each indicator in epilepsy rathippocampal slices detected with perfusion the ENT1selective inhibitorNBTI(100nM). Excitability changes of epilepsy rat hippocampal slices weredetected as action potentials and calculated the frequency. Moreover,evoked excitatory postsynaptic current and NMDA and AMPA receptormediated excitatory postsynaptic current were observed by calculating theiramplitude. In addition, spontaneous mini excitatory postsynaptic currentswere detected by calculating their frequency and amplitude.Results:1. NBTI decreases neuronal excitability in CA1pyramidal neurons ofepileptic rats: To validate the behavioral findings, we examined the firingproperties of hippocampal CA1pyramidal neurons in acutely isolated slices from epileptic rats24h after the onset of seizures. Bath application ofNBTI(100nM) significantly decreased the numbers of APs in epilepticneuron（sP <0.01）.It was also effective of NBTI on AP occurred in neuronstrated with different current injections (P <0.01).2. NBTI inhibition of evoked EPSC is mediated by adenosine A1receptor： Compared with the normal neurons(control), evoked EPSCsamplitude of epileptic neurons increased sinnificantly(P＜0.01). Bathapplication of adenosine (25μM) significantly reduced evoked EPSCsamplitude, while application of A1receptor inhibitor DPCPX partiallyreversed this effect (P＜0.01). Inhibition of EPSC by adenosine wasmimicked by NBTI (100nM), which was also reversed by DPCPX (P＜0.01). The inhibition of EPSC by adenosine was dose dependent (P＜0.01).At concentration of50M, the reduced EPSC by adenosine were furtherinhibited by NBTI (P＜0.01). However, when adenosine concentration wassaturated (100M), no further inhibitory effect of NBTI on EPSC wasobserved (P＞0.05).3. NBTI inhibits excitatory neurotransmission：Application of NBTIsignificantly reduced frequency but not amplitude of mEPSC in CA1neurons (P＜0.01). To further identify which receptor component ofglutamate, NMDAR or AMPAR, mediates NBTI effect on mEPSC, wemeasured evoked EPSC. Both NMDAR and AMPAR mediated componentswere increased significantly in epileptic hippocampal CA1neurons compared with normal neurons (P＜0.01). Application of NBTI led tosignificant inhibition of NMDAR and AMPAR mediated EPSCs (P＜0.01).Conclusion：1. NBTI possesses a strong inhibitory effect on epileptic hippocampalbrain slices discharge. The selective inhibition of ENT1function couldreduce epileptic rat hippocampal brain slices neurons excitability effectively.2. NBTI effect on EPSC was mediated by adenosine A1receptor. Theselective inhibition of ENT1function could reduce neurons excitabilitythrough adenosine A1receptor.3. NBTI selectively inhibits NMDAR，APMAR mediated EPSC, andmEPSC frequency，suggesting that the effect of NBTI may throughglutamate release in presynaptic structures. The selective inhibition ofENT1function could reduce glutamatergic neurotransmitter in presynapticstructures. PART FOUR: MECHANISM DETECTION OF ENT1ONEPILEPTIC SEIZURE IN VIVOObjective: To explore the potential mechanism of ENT1in epilepsy.Methods：The the ENT1selective inhibitor NBTI (50μM), its solvent（DMSO）and normal saline were injected into the rat hippocampus CA1region by three-dimensional positioning and microtubule.1．Adult male Sprague-Dawley rats (around200g) were randomlydivided into normal control group（n=5），epilepsy group (24h)（n=5），solvent control（vehicle） group（n=5） and NBTI group（n=5）. Byintraperitoneal injection of lithium chloride-pilocarpine, epilepsyexperimental animal model was established.2. The cAMP response element binding protein(CREB)phosphorylation levels in different groups were detected by weatern blot.Results: Compared with control group （0.44±0.06）， CREBphosphorylation levels increased24h after epilepsy（1.05±0.11）, whileNBTI injection in the hippocampus led to significantly decreased itsphosphorylation（0.47±0.07）(P＜0.01). In addition，there was no effect oflow concentration DMSO on CREB phosphorylation（1.09±0.12）（p＞0.05）.Conclusion：After inhibitting of ENT1by NBTI，the rat hippocampalCREB phosphorylation levels were significantly decreased, which indicatedthat CREB might be involved in the ENT1role in epilepsy. |
PDF Full Text Request